The immune system is formed by programmed recombination (V(D)J recombination). Mutations in Ku severely disrupt V(D)J recombination product formation and demonstrate defects in double--strand break repair (DSB repair). I propose to examine a cell line that is a Ku mutant in the Ku p70 subunit in a structure and function analysis to discriminate the role of different regions of this protein in V(D)J recombination and DSB repair. Ku p70 mutants will be examined for ability to restore V(D)J recombination, DSB repair, IRS, and DNA end binding defects. Mutants will also be constructed to test the role of Ku subunit interactions in Ku functions. DNA-PK phosphorylation sites of Ku p70 will be mapped and mutants of these sites will be studied in functional tests. These experiments are expected to delineate the significant features of the Ku p70 subunit in V(D)J recombination and DSB repair.